Mass spectrometry method for measuring vitamin b6 in body fluids

ABSTRACT

Provided are methods of detecting the presence or amount of the active form of vitamin B6, pyridoxal 5′-phosphate, in a body fluid sample using tandem mass spectrometry coupled with liquid chromatography.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/208,295, filed Aug. 11, 2011, which is a continuation applicationof U.S. application Ser. No. 11/763,380, filed Jun. 14, 2007, now U.S.Pat. No. 8,017,403, each of which is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The invention relates to the detection of the active form of vitamin B6,pyridoxal 5′-phosphate (PLP), by tandem mass spectrometry (MS-MS)coupled with liquid chromatography.

BACKGROUND OF THE INVENTION

Documents cited in this description are denoted numerically, inparentheses, by reference to a bibliography below. None of the citedreferences is admitted prior art.

Vitamin B6 is a water-soluble vitamin existing in seven forms:pyridoxine (PN), pyridoxine 5′-phosphate (PNP), pyridoxal (PL),pyridoxal 5′-phosphate (PLP), pyridoxamine (PM), pyridoxamine5′-phosphate (PMP), and 4-pyridoxic acid (PA). All forms except for PAare interconvertible in vivo. PN, PL and PM are natural compounds infood, which after absorption, are phosphorylated to an active form, toPNP, PLP, and PMP. PLP functions as a cofactor amino acid metabolism. PNis the form provided as vitamin B6 supplement. Under normal conditions,a 100 mg of vitamin B6 will produce a plasma peak in 2 hours with ahalf-life of 8 hours. Doses of over 25 mg produce little change inplasma PLP. PA is the ultimate metabolite of vitamin B6, which isexcreted in the urine.

Vitamin B6 plays an important role in the metabolism of amino acids,lipids, carbohydrates, and neurotransmitters. For instance, vitamin B6functions as a coenzyme for a number of key enzymes, such askynureninase, which catalyzes the synthesis of niacin from tryptophan;δ-aminolevulinic acid synthetase, which catalyzes heme synthesis; andglycogen phosphorylase, which catalyzes the release of glucose stored inthe muscle as glycogen. Vitamin B6 also participates in neurotransmittersynthesis and amino acid synthesis, as well as the catabolism ofhomocysteine to cystathionine and cystathionine to cysteine, whichinvolves a pathway closely related to increased homocysteine levels.

Vitamin B6 deficiency causes weakness, susceptibility to infection,sleeplessness, depression, dermatitis, glossitis, stomatitis andseizures. Chronic vitamin B6 deficiency may even cause severe nervecompression disorders. A low blood vitamin B6 level is considered a riskfactor for cardiovascular diseases. Although vitamin B6 deficiency israre as a low-intake malnutrition in the United States, it is commonlyassociated with other diseases, such as alcoholism, cirrhosis, kidneyfailure, and small intestinal malabsorption, etc. Therefore, it isimportant to detect the active form of vitamin B6, PLP, in plasma todetermine vitamin B6 deficiency or overdosage, thereby evaluatingwellness and monitoring treatment.

Conventionally, the level of aspartate aminotransferase is measured inred blood cells to diagnose vitamin B6 deficiency. This test, however,is a functional assay rather than a direct measurement of the PLP status(Ref. 1).

Clinical analysis of plasma PLP concentrations is a direct measurementof the active form of vitamin B6 in the blood. A number of publicationsreport successful detection of plasma PLP by high-performance liquidchromatographic assay (HPLC) (Refs. 2-8). Hachey et al. describedetermination of vitamin B6 in biological samples by mass spectrometry(Ref. 9). Liquid chromatography-tandem mass spectrometry can be used tomeasure vitamin B6 in human plasma (Ref. 10). Nevertheless, Rybak et al.report that there is an observed imprecision among the existingHPLC-based and enzymatic vitamin B6 assays (Ref. 24).

SUMMARY OF THE INVENTION

The present invention relates to detecting the presence or amount of theactive form of vitamin B6, pyridoxal 5′-phosphate (PLP), in a body fluidsample by tandem mass spectrometry (MS-MS) coupled with liquidchromatography. Preferably, the invention relates to detecting thepresence or amount of PLP in plasma.

In certain embodiments, a body fluid sample is a sample obtained from amammalian animal, such as a dog, cat, horse, etc. Particular preferredmammalian animals are primates, most preferably humans. Suitable samplesinclude blood, plasma, serum, urine, saliva, tears, and cerebrospinalfluid. Preferably, the sample is a plasma sample. Such samples may beobtained, for example, from a patient; that is, a living person in aclinical setting for diagnosis, prognosis, or treatment of a disease orcondition.

In one aspect, the invention provides a method for detecting thepresence or amount of PLP in a body fluid sample by tandem massspectrometry. The method include the steps of (i) purifying said sampleby liquid chromatography; (ii) generating a parent ion of PLP from thepurified sample; (iii) generating one or more daughter ions of theparent ion; and (iv) detecting the presence or amount of one or moreions generated in step (ii) or step (iii) or both, and relating thedetected ions to the presence or amount of PLP in the sample. In certainembodiments, the method is used to detect the presence or amount of PLPin plasma. In a preferred embodiment, the PLP in the sample is ionizedby electrospray.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the presence or amount of which is also detectedin the sample. In these embodiments, both PLP and the internal standardpresent in the sample are purified and then ionized to produce aplurality of ions detectable in a mass spectrometer operating inpositive ion mode, and one or more ions produced from each are detectedby mass spectrometry.

The term “operating in positive ion mode” refers to those massspectrometry methods where positive ions are detected. Similarly,“operating in negative ion mode” refers to those mass spectrometrymethods where negative ions are detected.

In preferred embodiments, the PLP ions detectable in a mass spectrometerinclude ions with a mass/charge ratio (m/z) of 248.03+/−1 m/z for theparent ion and 150.00+/−1 m/z for the daughter ion. The variation in them/z value observed by mass spectrometry for the parent and daughterion(s) generally ranges from 0.001 to 1 m/z unit of the atomic mass. Forexample, the variation in mass observed using different massspectrometers for identifying the parent 248.03 ion should be within 1m/z which is a range between 247.03 m/z and 249.03 m/z.

A preferred internal standard is 2-hydroxy-6-methylpyridine-3-carboxylicacid (Aldrich). In preferred embodiments, the internal standarddetectable in a mass spectrometer has a mass/charge ratio (m/z) of154.04+/−1 m/z for the parent ion and 136.00+/−1 m/z for the daughterion. An alternative internal standard for the assay is isotopicallylabeled vitamin B6.

If multiple daughter ions are monitored, the ion ratio variation of masstransitions of different daughter ions generated from the same parention should not exceed +/−25%, in accordance with the College of AmericanPathologists (CAP) standards. For example, if a compound generatesdaughter ions A and B from the same parent ion, the ion ratio is (theintensity of ion A divided by the intensity of ion B)×100.

In other preferred embodiments, the purifying step involves (a) applyinga body fluid sample to an extraction column; (b) washing the extractioncolumn under conditions whereby pyridoxal 5′-phosphate is retained bythe column; (c) eluting retained material comprising pyridoxal5′-phosphate from the extraction column; (d) applying the extractioncolumn-eluted material comprising pyridoxal 5′-phosphate to ananalytical column; and (e) eluting purified pyridoxal 5′-phosphate fromthe analytical column. In preferred embodiments, the extraction columnis a large particle extraction column, and the analytical column is aC-8 analytical column. In other embodiments, the extraction column is aturbulent flow liquid chromatography column, and more preferably a highturbulent flow liquid chromatography column (HTLC).

By “large particle” column, is meant a column containing an averageparticle diameter greater than about 35 μm. In the preferred embodiment,the extraction column contains particles of about 50 μm in diameter, andthe C-8 analytical column comprises particles of about 3.5 μm indiameter. As used in this context, the term “about” means±10%.Preferably, the extraction column is a mixed mode anion exchange polymercolumn with 0.5 (ID)×50 mm, 50μ particle size.

The term “analytical column” as used herein refers to a chromatographycolumn having sufficient chromatographic “plates” to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such a columnis often distinguished from an “extraction column,” which has thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. A preferred analytical column is an HPLC column.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram of PLP in EDTA-plasma upon tandem massspectrometry analysis at the following integration parameters:

m/z values of parent/daughter ions: 248.03/150.00 amu;

SW: 500.00;

View width (minutes): 3.75RT reference (RTR): Yes;Minimum peak height (MPH, signal/noise ratio): 3.0;

Smooth: 5;

Peak detection algorithm (PDA): genesis.The chromatogram is produced by the manufacturer-provided software (Ariaversion 1.5, Cohesive Technologies) using the peak intensity (areacounts) from the mass spectrometry. As shown in the FIG. 1, the highestpeak of PLP appears at an estimated retention time of 0.75 minute.

FIG. 2 is a chromatogram of the internal standard,2-hydroxy-6-methylpyridine-3-carboxylic acid, upon tandem massspectrometry analysis at the following integration parameters:

m/z values of parent/daughter ions: 154.04/136.00 amu;

SW: 50.00;

View width (minutes): 3.75RT reference (RTR): Yes;Minimum peak height (MPH, signal/noise ratio): 3.0;

Smooth: 5;

Peak detection algorithm (PDA): genesis.The internal standard is added to each sample in equal amount for eachprocess. As shown in the FIG. 1, the highest peak of2-hydroxy-6-methylpyridine-3-carboxylic acid appears at an estimatedretention time of 3.16 minute.

FIG. 3 depicts the typical standard curve of PLP. The software uses peakarea ratio of PLP over its internal standard for data reduction. Thepeak areas of internal standard are consistent (within +/−30%) for allthe samples tested, and the concentration levels of PLP correspond tothe peak area ratios of PLP vs. internal standard. The peak area ratioobtained from MS/MS is plotted against various known PLP concentrationsbetween 2 ng/mL and 250 ng/mL to obtain the standard curve. A blankplasma or serum as zero standard is included. Linear regression with1/×weighting is used for data reduction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes methods for detecting the presence oramount of the active form of vitamin B6, PLP, in a sample. The methodsutilize liquid chromatography to perform an initial purification of PLPfollowed by tandem mass spectrometry (MS/MS), thereby providing ahigh-throughput assay system for detecting and quantifying PLP in aliquid sample. The assay offers enhanced specificity and is accomplishedin less time and with less sample preparation than required by othervitamin B6 assays. In various embodiments the methods of the inventionaccurately quantify PLP in samples where it is present in the rangebetween 2 ng/mL and 250 ng/mL, while the method is sufficientlysensitive to detect PLP at a concentration as low as 0.96 ng/ml.

Sample Purification by HTLC/HPLC

Preferably, PLP is initially purified by high turbulence-flow liquidchromatography (HTLC) and high performance liquid chromatography (HPLC)prior to mass spectrometry. “Purification” in this context does notrefer to removing all materials from the sample other than theanalyte(s) of interest. Instead, purification refers to a procedure thatenriches the amount of one or more analytes of interest relative to oneor more other components of the sample. In preferred embodiments,purification can be used to remove one or more interfering substances,e.g., one or more substances that would interfere with detection of ananalyte ion by mass spectrometry.

HTLC is a form of chromatography that utilizes turbulent flow of thematerial being assayed through the column packing as the basis forperforming the separation. HTLC has been applied in the preparation ofsamples containing two unnamed drugs prior to analysis by massspectrometry (Refs. 11-15). Persons of ordinary skill in the artunderstand “turbulent flow.” When fluid flows slowly and smoothly, theflow is called “laminar flow.” In laminar flow the motion of theparticles of fluid is orderly with particles moving generally instraight lines. In contrast, at faster velocities, the inertia of thewater overcomes fluid frictional forces and turbulent flow results.Fluid not in contact with the irregular boundary “outruns” that slowedby friction or deflected by an uneven surface. When a fluid is flowingturbulently, it flows in eddies and whirls (or vortices), with more“drag” than when the flow is laminar. Many references are available forassisting in determining when fluid flow is laminar or turbulent (Refs.16 & 17).

Traditional HPLC analysis relies on the chemical interactions betweensample components and column packings, in which laminar flow of thesample through the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a partition process. In contrast, it isbelieved that “turbulent flow,” such as that provided by HTLC columnsand methods, may enhance the rate of mass transfer, thereby improvingthe separation characteristics provided by the separation system. HTLCcolumns separate components by means of high chromatographic flow ratesthrough a packed column containing rigid particles. By employing highflow rates (e.g., 3-4 ml/min), turbulent flow occurs in the column thatcauses nearly complete interaction between the stationary phase and theanalytes. An additional advantage of HTLC columns is that themacromolecular build-up associated with biological fluid matrices isavoided since the high molecular weight species are not retained underthe turbulent flow conditions.

Because HTLC method can combine multiple separations in one automatedprocedure, it not only lessens the need for lengthy sample preparationand operates at a significantly greater speed, but also eliminates theopportunity for operator error by minimizing operator involvement duringthe purification. Such a method also achieves a separation performancesuperior to laminar flow (HPLC) chromatography. HTLC allows for directinjection of biological samples (plasma, urine, etc.). This is difficultto achieve in traditional forms of chromatography because denaturedproteins and other biological debris quickly block the separationcolumns.

Additionally, the commercial availability of HTLC apparatuses thatpermit multiplexing of columns and direct integration with MSinstruments makes such instruments particularly well suited tohigh-throughput applications.

During chromatography, the separation of materials is effected byvariables such as choice of eluant (also known as a “mobile phase”),choice of gradient elution and the gradient conditions, temperature,etc.

The present invention applies a complex gradient for HTLC and HPLCchromatography. More specifically, the gradient starts with pumping 100%deionized water at 0.4 mL/minute on the HTLC column and 5% acetic acidat 0.4 mL/minute on the HPLC column for 60 seconds. The sample isinjected at the same time as the gradient begins. Subsequently, the HTLCcolumn is washed with 100% deionized water at a flow rate of 1.5mL/minute for 50 seconds, followed by 5% aqueous acetic acid at the samerate for 40 seconds. The extract is eluted from the HTLC column to theHPLC column with 5% aqueous acetic acid at a flow rate of 0.4 mL/minutefor 200 seconds, starting at 2 minutes calculated from sample injection.Afterwards, the elution from the HPLC column is driven by a gradient of5% aqueous acetic acid/acetonitrile (45 to 15%/55 to 85%) at a flow rateof 0.4 mL/minute. The reminder of the gradient time is for columncleaning and re-equilibration with deionized water, 5% aqueous aceticacid and acetonitrile. The total duration for chromatograph is 9.0minutes, with 4.3-minute data window for each sample. Therefore, twosamples can be analyzed within the 9-minute method time when the dualchannels are operated with overlapping gradients.

In certain embodiments, an analyte may be purified by applying a bodyfluid sample to a column under conditions where the analyte of interestis reversibly retained by the column packing material, while one or moreother materials are not retained. In these embodiments, a first mobilephase condition can be employed where the analyte of interest isretained by the column, and a second mobile phase condition cansubsequently be employed to remove retained material from the column,once the non-retained materials are washed through. The second mobilephase may be phased in gradually, usually under computer controldirecting the composition of mobile phase over time, or by an immediatechange in the mobile phase. The retained materials may also be removedfrom the column by “backflushing” the column, or reversing the directionof flow of the mobile phase. This may be particularly convenient formaterial that is retained at the top of the column. Alternatively, ananalyte may be purified by applying a sample to a column under mobilephase conditions where the analyte of interest elutes at a differentialrate in comparison to one or more other materials. As discussed above,such procedures may enrich the amount of one or more analytes ofinterest relative to one or more other components of the sample.

In a preferred embodiment, PLP is initially purified by liquidchromatography, which involves the steps of (a) applying a body fluidsample to an extraction column; (b) washing the extraction column underconditions whereby pyridoxal 5′-phosphate is retained by the column; (c)eluting retained material comprising pyridoxal 5′-phosphate from theextraction column; (d) applying the extraction column eluted materialcomprising pyridoxal 5′-phosphate to an analytical column; and (e)eluting purified pyridoxal 5′-phosphate from the analytical column.Preferably, the extraction column is an HTLC column and the analyticalcolumn is an HPLC column.

In various embodiments, one or more of the purification and/or analysissteps can be performed in an online, automated fashion. For example, inone embodiment, the purification steps (a)-(e) are performed in anonline, automated fashion. In another embodiment, the steps of massspectrometry are performed online following steps (a)-(e). The term“online, automated fashion” as used herein refers to steps performedwithout the need for operator intervention. For example, by carefulselection of valves and connector plumbing, two or more chromatographycolumns can be connected as needed such that material is passed from oneto the next without the need for any manual steps. In preferredembodiments, the selection of valves and plumbing is controlled by acomputer pre-programmed to perform the necessary steps. Most preferably,the chromatography system is also connected in such an online fashion tothe detector system, e.g., an MS system. Thus, an operator may place atray of samples in an autosampler, and the remaining operations areperformed under computer control, resulting in purification and analysisof all samples selected.

In contrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator after the sample is loadedonto the first column. Thus, if samples are subjected to precipitation,and the supernatants are then manually loaded into an autosampler, theprecipitation and loading steps are off-line from the subsequent steps.

Sample Analysis by MS/MS

Purified sample is subjected to ionization before analyzed by MS/MS. Asused herein, the term “ionization” refers to the process of generatingan analyte ion having a net electrical charge equal to one or moreelectron units. Negative ions are those having a net negative charge ofone or more electron units, while positive ions are those having a netpositive charge of one or more electron units.

The term “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”) (Refs. 18-23).

The ions may be detected using several detection modes. For example,selected ions may be detected using a selective ion monitoring mode(SIM), or alternatively, ions may be detected using a scanning mode,e.g., multiple reaction monitoring (MRM) or selected reaction monitoring(SRM).

Preferably, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and m/z. The voltage and amplitude can beselected so that only ions having a particular m/z travel the length ofthe quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments can act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

“Tandem mass spectrometry,” or “MS/MS” is employed to enhance theresolution of the MS technique. In tandem mass spectrometry, a parention (a.k.a. precursor ion) generated from a molecule of interest may befiltered in an MS instrument, and the parent ion subsequently fragmentedto yield one or more daughter ions (a.k.a. fragment or product ions)that are then analyzed in a second MS procedure.

Collision-induced dissociation (“CID”) is often used to generate thedaughter ions for further detection. In CID, parent ions gain energythrough collisions with an inert gas, such as argon, and subsequentlyfragmented by a process referred to as “unimolecular decomposition.”Sufficient energy must be deposited in the parent ion so that certainbonds within the ion can be broken due to increased vibrational energy.

By careful selection of parent ions, only ions produced by certainanalytes of interest are passed to the fragmentation chamber to generatethe daughter ions. Because both the parent and daughter ions areproduced in a reproducible fashion under a given set ofionization/fragmentation conditions, the MS/MS technique can provide anextremely powerful analytical tool. For example, the combination offiltration/fragmentation can be used to eliminate interferingsubstances, and can be particularly useful in complex samples, such asbiological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each m/z over a given range (e.g., 100 to900 amu). The results of an analyte assay, that is, a mass spectrum, canbe related to the amount of the analyte in the original sample bynumerous methods known in the art. For example, given that sampling andanalysis parameters are carefully controlled, the relative abundance ofa given ion can be compared to a table that converts that relativeabundance to an absolute amount of the original molecule. Alternatively,molecular standards can be run with the samples, and a standard curveconstructed based on ions generated from those standards. Using such astandard curve, the relative abundance of a given ion can be convertedinto an absolute amount of the original molecule. In certain preferredembodiments, an internal standard is used to generate a standard curvefor calculating the quantity of PLP. Numerous other methods for relatingthe presence or amount of an ion to the presence or amount of theoriginal molecule are well known to those of ordinary skill in the art.

The skilled artisan will understand that the choice of ionization methodcan be determined based on the analyte to be measured, type of sample,the type of detector, the choice of positive versus negative mode, etc.Ions can be produced using a variety of methods including, but notlimited to, electron ionization, chemical ionization, fast atombombardment, field desorption, and matrix-assisted laser desorptionionization (MALDI), surface enhanced laser desorption ionization(SELDI), photon ionization, electrospray ionization, and inductivelycoupled plasma. Electrospray ionization is a preferred ionizationmethod. The term “electrospray ionization,” or “ESI,” as used hereinrefers to methods in which a solution is passed along a short length ofcapillary tube, to the end of which is applied a high positive ornegative electric potential. Solution reaching the end of the tube, isvaporized (nebulized) into a jet or spray of very small droplets ofsolution in solvent vapor. This mist of droplets flows through anevaporation chamber which is heated slightly to prevent condensation andto evaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

The concentration of PLP in the plasma between 3 and 35 ng/mLestablished by radioenzymatic assay (REA) for a child of 2-17 years old,or between 3 and 26 ng/mL established by REA for an adult of 18 years orolder, is within the normal range. The adult reference values obtainedby REA are correlated well with those obtained by the LC-MS-MS method ofthe invention. A slope of 0.9745 is observed, i.e. the values obtainedby the LC-MS-MS method are 2.6% higher than the REA valuesstatistically.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the invention.

Example 1 Sample Preparation

A. Plasma Sample

The blood is collected in EDTA from a subject following an overnightfast. The subject should be restricted from alcohol and vitaminsconsumption for 24 hours before sample collection. Plasma should beseparated from cells immediately upon collection of blood or within 6hours from the whole blood that is kept refrigerated and protected fromlight.

The cells are separated by centrifugation at 2-8° C. at 2,200-2,500 rpm,800-1000 g for 5-10 minutes. Then the plasma is transferred to adark-brown polypropylene or polyethylene transport tube to protect itfrom light. Alternately, a neutral color polypropylene or polyethylenetube can be used if it is wrapped in aluminum foil. Freeze the tube at−10 to −30° C. The samples are acceptable if they are kept at −10 to−30° C. for 6 days, or at −60 to −80° C. for 6 weeks.

B. Internal Standard Stock and Working Solutions

An internal standard stock solution of 1.0 mg/mL2-hydroxy-6-methylpyridine-3-carboxylic acid (Aldrich, Catalog No.H43008) is prepared in de-ionized water. An internal standard workingsolution of 1.25 μg/mL is prepared in 0.1% formic acid from the stocksolution.

C. PLP Stock and Working Solutions

All PLP solutions should be protected from exposure to light.

PLP (Sigma, Catalog No. P9255) stock solution of 1.0 mg/mL is preparedin de-ionized water. Then PLP stock solution of 5 μg/mL is prepared in0.1% formic acid from the 1.0 mg/mL stock. Intermediate PLP standardsolution of 500 ng/mL is prepared from the 5 μg/mL intermediate PLPstock solution by dilution with PLP-free plasma/serum (Biocell Labs,Carson, Calif., Catalog No. 1131-00). The analyte-stripped, delipidizedand defibrinated plasma/serum from Biocell is checked before use to besure of PLP-free.

PLP working standards are prepared by serial dilution of the 500 ng/mLPLP standard solution in PLP-free plasma/serum (Biocell Labs, Carson,Calif., Catalog No. 1131-00), as indicated in the table below. Theseworking standards are used to generate the PLP standard curveillustrated in FIG. 3.

Biocell Target Concentration Serum Standard Plasma/Serum (ng/mL) (uL)(uL) Blank  0 200 S1 = 2 200 of S2 200 S2 = 3.91 200 of S3 300 S3 = 7.82200 of S4 200 S4 = 15.63 200 of S5 200 S5 = 31.25 200 of S6 200 S6 =62.5 200 of S7 200 S7 = 125 200 of S8 200 S8 = 250 200 of 200intermediate PLP standard solution

Example 2 Sample Purification

The process from sample injection to the column to chromatogram is anintegrated process involving HTLC for online extraction, purificationand supplying the desired analytes to the HPLC column, HPLC foranalytical separation of the analytes and mass spectrometry for compoundidentification and acquiring specific mass to charge ratio for eachanalyte. As unbound and unwanted debris is swept through the extractioncolumn at high velocity, the PLP, as well as the internal standard, iscaptured and concentrated in the column. The HTLC extraction column isthen backflushed and the sample is loaded onto an HPLC analyticalcolumn. The analytical column is then subjected to an elution gradient.The columns are online and allow for the chromatographic separation ofthe components of interest. The purification process is described infurther details below.

200 μL of plasma samples, including standard, control, blank andunknown, are mixed with 0.6 mL of internal standard working solution in0.1% formic acid before online extraction. Pour the samples individuallyinto a 96-well plate and cover. Place the 96-well plate into autosamplercooling unit. Then 10±2 μl of the sample is injected into the CohesiveTX-4 HTLC system.

The HTLC system is logically divided into two functions: 1) Solid phaseextraction using a large particle size (e.g., 50 μm) packed column and2) HPLC chromatography using a binary gradient and a 3.5 μm reversephase analytical column. In this example, a Cohesive Cyclone® Max 0.5×50mm column (Cohesive Technologies, Catalog No. 952980) was used forextraction. The extraction column is a mixed mode anion exchange polymercolumn with 0.5 (ID)×50 mm, 50μ particle size. The extraction columnretains PLP, the internal standard and smaller molecules, while allowinglarger molecules and electrolytes be eluted out of the column.

In the chromatography process, the gradient was started with pumping100% deionized water at 0.4 mL/minute on the HTLC column and 5% aceticacid at 0.4 mL/minute on the HPLC column for 60 seconds. The sample wasinjected at the same time as the gradient began. Subsequently, the HTLCcolumn was washed with 100% deionized water for 50 seconds, followed by5% aqueous acetic acid for 40 seconds, at a high (about 1.5 mL/minute)flow rate. The high flow rate creates turbulence inside the extractioncolumn. This turbulence ensures optimized binding of PLP to the largeparticles in the column and the passage of residual protein and debristo waste.

After the loading and washing steps, the sample was eluted off of theHTLC extraction column and transferred to the analytical HPLC column,with 5% aqueous acetic acid at a flow rate of 0.4 mL/minute for 200seconds, starting at 2 minutes calculated from sample injection. TheHPLC column is a C-8 column with a particle size of 3.5 μm. Such HPLCcolumns are commercially available. The column used in this example wasZorbax SB-C8 2.1×150 mm column (Agilent Technologies, Catalog No.830990-906).

The elution from the HPLC column was driven by a binary gradient of 5%aqueous acetic acid/acetonitrile (45 to 15%/55 to 85%) at a flow rate of0.4 mL/minute. The reminder of the gradient time is for column cleaningand re-equilibration with deionized water, 5% aqueous acetic acid andacetonitrile. The total duration for chromatograph is 9.0 minutes, with4.3-minute data window for each sample. Therefore, two samples can beanalyzed within the 9-minute method time when the dual channels areoperated with overlapping gradients.

The selected parameters for HTLC were as follows:

Injection volume: 10±2 μLAutosampler tray temperature: 5±2° C.Switch valve loop size: 200 μL

For the loading pumps, loading aqueous solution A is 5% acetic acid,loading organic solution B is 100% acetonitrile, and loading aqueoussolution C is 100% water. For eluting pumps, eluting solution A is 5%acetic acid and eluting solution B is 100% acetonitrile. The totalmethod duration is 9 minutes with 4.33 minutes data window foracquisition.

After elution, PLP was separated from other analytes in the sample. Theseparated PLP was then transferred to the MS/MS for quantification.

Example 3 Sample Analysis

The detection was accomplished by electrospray triplequad MS-MS system(Thermo Finnigan TSQ Quantum Ultra). The flow of liquid solvent from theanalytical column entered the heated nebulizer interface of the MS/MSanalyzer. The solvent/analyte mixture was first converted to vapor inthe heated tubing of the interface. The analytes, contained in thenebulized solvent, were ionized and a positive charge added by thecorona discharge needle of the interface, which applies a large voltageto the nebulized solvent/analyte mixture. The ions passed through theorifice of the instrument and entered the first quadrapole. Quadrapoles1 and 3 (Q1 and Q3) were the mass filters, allowing selection of ionsbased on their mass to charge ratio (m/z). Quadrapole 2 (Q2) was thecollision cell, where ions were fragmented by collision with argonmolecules.

The first quadrapole of the MS/MS (Q1) selected for PLP with an m/z of248.03 or the internal standard of 154.04. Ions with these m/z valuespassed to the collision chamber (Q2), while ions with any other m/zcollided with the sides of the quadrapole and were destroyed. Ionsentering Q2 collided with neutral gas molecules. This process is calledCollision-Induced Dissociation (CID). The CID gas used in this examplewas argon. The daughter ions generated were passed into quadrapole 3(Q3), where the daughter ions of PLP (m/z 150.00) or those of theinternal standard (m/z 136.00) were selected for, while other ions werescreened out. The selected daughter ions were collected by the detector.Quantification is based on peak area ratio of analytes over the internalstandard acquired by selective reaction monitoring (SRM) in positivemode.

Selected MS/MS parameters were:

-   Scan type: positive ion SRM-   Run time: 4.5±0.2 minutes-   MS/MS transitions: PLP (248.03 parent ion to 150.00 daughter ion)    internal standard (154.04 parent ion to 136.00 daughter ion)    MS common parameters:

spray voltage: 3200

sheath gas: 50

aux gas: 10

capillary temperature: 350° C.

tube lens offset: 63

collision energy: 10

scan width: 0.002

scan time: 0.1

Q1 resolution: 0.2

Q2 resolution: 0.7

As ions collide with the detector, they produce a pulse of electrons.The pulse was converted to a digital signal, which was counted toprovide an ion count. The acquired data was relayed to the computer,which plotted counts of the ions collected vs. time. Heights of thepeaks generated were computer-measured, response factors were generatedfrom calibration material, and PLP thereby quantified in the sample.

Example 4 Determination of Sample Concentration

Calculations of the unknown concentration are based on peak area ratiosof PLP over the internal standard against a standard curve using LCquansoftware in Xcalibur (Thermo Finnigan). The peak area ratios of thedaughter ions for PLP and the internal standard were used forcalculations.

First, a calibration standard curve is plotted using 8 PLP standards, at2 ng/mL, 3.91 ng/mL, 7.82 ng/mL, 15.63 ng/mL, 31.25 ng/mL, 62.5 ng/mL,125 ng/mL and 250 ng/mL, respectively. A blank plasma/serum as zerostandard is usually included each time. The software uses peak arearatio of PLP over the internal standard for data reduction. The mathmodel of linear regression with 1/× weighting is used for datareduction. The peak area ratios of PLP vs. the internal standard isplotted against the known PLP concentrations to obtain the standardcurve. An acceptable calibration curve must have a correlationcoefficient (R²) of 0.9950 or better.

To determine the concentration of PLP in a sample, the peak area ratioof PLP in the sample over the internal standard is established by theLC/MS/MS method, and then integrated to the standard curve by LCquansoftware.

CITED PUBLICATIONS

Each of the following publications as well as each publication citedabove is incorporated herein, in its entirety, by reference.

-   1. Talwar et al., “Pyridoxal phosphate decreases in plasma but not    erythrocytes during systemic inflammatory response,” Clinical    Chemistry 49: 515-518, 2003-   2. Bisp et al., “Determination of vitamin B6 vitamers and pyridoxic    acid in plasma: development an devaluation of a high-performance    liquid chromatographic assay,” Anal. Biochem. 305: 82-89, 2002-   3. Bailey et al., “High performance liquid chromatography method for    the determination of pyridoxal-5-phosphate in human plasma: how    appropriate are cut-off values for vitamin B6 deficiency?” Eur. J.    Clin. Nutr. 53: 448-455, 1999-   4. Kimura et al., “Highly sensitive and simple liquid    chromatographic determination in plasma of B6 vitamers, especially    pyridoxal 5′-phosphate,” J. Chromatogr A. 722: 296-301, 1996-   5. Edwards et al., “A simple liquid-chromatographic method for    measuring vitamin B6 compounds in plasma,” Clin. Chem. 35: 241-245,    1989-   6. Botticher et al., “A new HPLC-method for the simultaneous    determination of B1-, B2- and B6-vitamers in serum and whole blood,”    Int. J. Vitam. Nutr. Res. 57: 273-278, 1987-   7. Ubbink et al., “Stability of pyridoxal-5-phosphate semicarbazone:    applications in plasma vitamin B6 analysis and population surveys of    vitamin B6 nutritional status,” J. Chromatogr. 342: 277-284, 1985-   8. Schrijver et al., “Semi-automated fluorometric determination of    pyridoxal-5′-phosphate (vitamin B6) in whole blood by    high-performance liquid chromatography (HPLC),” Int. J. Vitam. Nutr.    Res., 51: 216-222, 1981-   9. Hachey et al., “Quantitation of vitamin B6 in biological samples    by isotope dilution mass spectrometry,” Anal. Biochem. 151: 159-168,    1985-   10. Midttun et al., “Multianalyte quantification of vitamin B6 and    B2 species in the nanomolar range in human plasma by liquid    chromatography-tandem mass spectrometry,” Clin. Chem., 51:    1206-1216, 2005-   11. Zimmer et al., J. Chromatogr. A 854: 23-35 (1999)-   12. U.S. Pat. No. 5,968,367-   13. U.S. Pat. No. 5,919,368-   14. U.S. Pat. No. 5,795,469-   15. U.S. Pat. No. 5,772,874-   16. P. S. Bernard & J. M. Wallace, “Turbulent Flow Analysis:    Measurement and Prediction,” John Wiley & Sons, Inc. (2000)-   17. Jean Mathieu & Julian Scott, “An Introduction to Turbulent    Flow,” Cambridge University Press (2001)-   18. U.S. Pat. No. 6,204,500-   19. U.S. Pat. No. 6,107,623-   20. U.S. Pat. No. 6,268,144-   21. U.S. Pat. No. 6,124,137-   22. Wright et al., “Proteinchip surface enhanced laser    desorption/ionization (SELDI) mass spectrometry: a novel protein    biochip technology for detection of prostate cancer biomarkers in    complex protein mixtures,” Prostate Cancer and Prostatic Diseases 2:    264-76 (1999)-   23. Merchant and Weinberger, “Recent advancements in    surface-enhanced laser desorption/ionization-time of flight-mass    spectrometry,” Electrophoresis 21: 1164-67 (2000)-   24. Rybak et al., “Clinical vitamin B6 analysis: an interlaboratory    comparison of pyridoxal 5′-phosphate measurements in serum,”    Clinical Chemistry 51:1223-1231 (2005)

What is claimed is:
 1. A method for detecting the presence or amount ofpyridoxal 5′-phosphate in a body fluid sample by tandem massspectrometry, comprising: (i) purifying said sample with an extractioncolumn and an analytical column for chromatographic separation; (ii)generating a parent ion of said pyridoxal 5′-phosphate from saidpurified sample; (iii) generating one or more daughter ions of saidparent ion; and (iv) detecting the presence or amount of one or moresaid ions generated in step (ii) or step (iii) or both, and relating thedetected ions to the presence or amount of said pyridoxal 5′-phosphatein said sample.
 2. The method of claim 1, wherein the extraction columnis a mixed mode anion exchange polymer column, and the analytical columnis a C-8 analytical column.
 3. The method of claim 2, wherein theextraction column comprises particles of about 50 μm, and the analyticalcolumn comprises particles of about 3.5 μm.
 4. The method of claim 1,wherein steps (i)-(iv) are performed in an online automated fashion. 5.The method of claim 1, wherein said parent ion has a mass/charge ratioof 248.03+/−1.
 6. The method of claim 5, wherein said one or moredaughter ions comprise a daughter ion with a mass/charge ratio of150.00+/−1.
 7. The method of claim 1, wherein said sample comprisesplasma.
 8. The method of claim 1, wherein step (ii) is performed byelectrospray ionization.
 9. The method of claim 1, wherein step (iii) isperformed by Collision-Induced Dissociation using a neutral gas.
 10. Themethod of claim 1, wherein an internal standard is added to the bodyfluid sample prior to step (i).
 11. The method of claim 1, wherein saidanalytical column is a high performance liquid chromatography column.12. The method of claim 1, further comprising protein precipitationprior to the purification step.